Epithelial sodium channels (ENaC) are members of the ENaC/degenerin family of ion channels that includes acid-sensing ion channels (ASIC) in mammals, mechanosensitive degenerin channels in worms, and FMRF-amide peptide-gated channels in mollusks (Kellenger, S. and Schild, L. (2002) Physiol. Rev. 82:735-767). ENaC mediates amiloride-sensitive apical membrane Na+ transport across high resistance epithelia in numerous tissues including kidney, colon, and lung.
ENaC is known to be a heterotrimeric channel comprised of α, β, and γ subunits. This heterotrimeric channel has been hypothesized to be involved in human salty taste perception. Previously, assays have been developed by the present assignee using ENaC sequences to identify compounds that modulate the delta beta gamma and alpha beta gamma human ENaC to examine if these compounds will potentially modulate human salty taste perception. Also, these compounds potentially may be used to treat human pathologies involving aberrant ENaC function.
Unlike other mammals, amiloride only slightly reduces the intensity of sodium chloride taste, i.e., by about 15-20% when used at concentrations that specifically modulate ENaC function. Experiments conducted by the inventors have shown that amiloride, or the more potent amiloride derivative phenamil did not elicit a significant effect on perceived human salt intensity when tested at levels 300-fold (for amiloride) and 3000-fold (for benzamil) above IC50 values for alpha beta gamma ENaC in oocytes (equivalent to 10-fold for amiloride and 100-fold for benzamil over IC50 values for delta beta gamma ENaC in oocytes). Thus, additional non-ENaC genes are likely involved in human taste and other biological functions.
This invention relates to fluorescent assays that identify compounds that modulate (enhance) ENaC in test cells which express a functional delta or alpha sodium epithelial channel, preferably the human alpha or delta ENaC, and the use thereof e.g., to modulate human umami or salty taste perception or other biological functions involving delta ENaC or alpha ENaC such as described herein. More specifically the invention provides improved fluorimetric assays and conditions for use therein that contact test cells which preferably express the three alpha or delta ENaC subunits with specific membrane potential or ion sensitive dyes under conditions that result in a profound enhancement in fluorescence signal relative to prior assays. As shown infra, it has been surprisingly discovered that when ENaC expressing cells are loaded with membrane potential dye in the presence of a sufficient amount of sodium and for a sufficient time and the test cells thereafter contacted with at least one putative ENaC enhancer that a substantially enhanced fluorimetric signal is generated than prior assays wherein sodium was absent or was at most 17.5 mM during membrane potential dye loading and prior to contacting the test cells with the putative enhancer. Thus this invention should provide for the identification of ENaC enhancers undetectable in prior assays.
Based thereon, improved assays have been developed which are disclosed herein to identify compounds that modulate the alpha and delta human ENaC. These compounds will be useful in modulating human taste, e.g., umami or human salty taste perception. Also, as described above these compounds may be used to treat human pathologies involving ENaC function.
Particularly Also the invention relates to the use of the subject novel assays for identifying compounds that enhance or inhibit delta ENaC can selectively modulate umami taste function and response to umami tastants.
Further the invention relates to use of the subject improved assays for identifying compounds that enhance or inhibit delta ENaC which can modulate umami taste cell development and apoptosis.
Further, the invention relates to the use of the subject improved assays to identify compounds that enhance or inhibit delta ENaC can modulate the function of any cell expressing the umami taste receptor including by way of example cells in the gastrointestinal tract such as enteroendocrine cells that regulate gastric motility and peptide secretion or digestion (e.g., GLP-1, glucagon-like peptide 1, GIP (gastric inhibitory peptide), secretin, amylase, cholecystokinin and the like).
Further, the invention relates to the use of the subject assays to identify compounds that enhance or inhibit alpha ENaC which can modulate sweet, bitter, umami and sour taste function and responses to sweet, bitter, umami, and sour tastants. This is predicated in part on the observation that salt (sodium) makes food taste better which may relate to sodium ions flowing through alpha ENaC ion channels and depolarizing sweet, bitter, umami, and sour taste cells, thereby leading to their activation and transmission of signals to the brain indicating enhanced sweet, bitter, umami, and sour taste perception.
Also the invention relates to the use of the subject assays to identify compounds that bind to or which modulate delta ENaC activity which may be used to treat or prevent pathological conditions involving digestive function. These conditions include by way of example functional dyspepsia (bad digestion) and other dyspepsias which may or may not be ulcer derived or related and may involve different areas of the digestive tract such as the upper abdominal tract, the mid-abdominal tract or the lower abdominal tract.
Also the invention relates to the discovery that because delta ENaC is expressed in taste cells such as umami cells the invention relates to the use of the subject assays to identify compounds that may be used to treat or prevent pathological conditions involving gastrointestinal hormones involved with digestion or hunger such as gastrin, secretin, cholecystokin, gastric inhibitory peptide, glucagon-like peptide 1, amylase, or ghrelin, leptin and the like. These compounds potentially may be used to suppress or induce hunger or to modulate digestion in subjects in need thereof.
Also because delta ENaC is expressed in taste cells such as umami cells the invention also relates to the use of the subject assays to identify compounds that bind to or modulate the activity of delta ENaC which compounds potentially may be used to treat or prevent pathological or chronic inflammatory or autoimmune gastrointestinal conditions such as Crohn's disease, inflammatory bowel syndrome (IBD), celiac disease, ulcerative colitis, diverticulitis, gastritis, reflux esophagitis, and the like. These compounds potentially may be used to treat or prevent autoimmune or inflammatory diseases affecting the digestive system.
Also, because delta ENaC is expressed in taste cells, e.g., umami cells the invention further relates to the use of the subject assays which use delta ENaC or cells which express delta ENaC such as taste cells, e.g., gastrointestinal or oral cavity derived cells in screening assays to identify compounds that bind to or modulate delta ENaC which potentially may be used to modulate gastric reflux and diseases or conditions associated therewith such as gastroesophageal reflux disease, heartburn, Barrett's esophagus, and esophagitis.
Also because delta ENaC is expressed in taste cells, e.g., umami cells the invention further relates to the use of the subject assays which use delta ENaC or cells which express delta ENaC in screening assays to identify compounds that bind to or which modulate delta ENaC activity and which therefore potentially may be used to treat or prevent cancers or malignancies associated with the digestive system such as by way of example cancers of the tongue, oral cavity, stomach, esophagus, small or large intestine, anus, pancreas, gall bladder, liver, or colon.
Also because delta ENaC is expressed in taste cells, e.g., umami cells the invention further relates to the use of the subject assays that use delta ENaC or cells which express delta ENaC in screening assays to identify compounds that bind to or which modulate the activity of delta ENaC which compounds potentially my be use to treat or prevent appetite dysfunction and conditions associated therewith such as anorexia, bulimia, and cachexias associated therewith.